Project Summary Sexual reproduction requires a germ line and mammals craft the beginning of their germ line early in development by secreted signaling molecules. This mechanism in vivo remains obscure, largely from the technical limitations of experimenting on embryos developing in utero. Germ line induction in a human embryo is even more remote and errors in the process are associated with a lack of germ line (infertility) and mis- placed specification of germ line cells (teratomas). How the primordial germ cells form by cell-cell interactions during early development is the focus of this application and makes use of a sister group to chordates ? the sea star embryo. While not a common organism for biomedical research, the sea star model system has many experimental and strategic attractions for revealing this process. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germ line, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop- mRNA-seq, optogenetic controls) and they respond well to complementary gene perturbation approaches (CRISPR, morpholino (MASO), and small molecule perturbations. The existing genomic and reagent resources for the sea star, coupled with the tractable experimental characteristics of the sea star embryo, yields a unique and surrogate system for understanding mammalian germ line induction. The approaches documented in this application will accomplish three main goals to advance the field in transformative ways. 1) Three signaling pathways are prioritized for integrative stimulating and repressing activities during a brief window in early development. 2) The mechanism of germ cell fate restriction is interrogated by a restriction map - germ cell factors are retained in the future germ line, whereas somatic factors are rapidly restricted from the same field. 3) Commitment to the germ line is hypothesized to include rapid insulation from local embryonic signaling and instead transitions to a committed cell type with a unique transcriptome, and response to signal activity. Single cell mRNA drop-sequencing and optogenetic control over signaling at single cell resolution are used by taking full advantage of the optical clarity and accessibility of the embryo. Overall, these accomplished goals will provide direct translation for understanding inductive germ line formation in mammals, and will reveal mechanistic explanations for germ cell-less conditions leading to human infertility, and mis-expression of germ cells leading to germ line tumors.